Plant shoot growth occurs mainly from the continual activity of the apical meristem. The biochemical and cytological events associated with apical growth are of paramount importance in understanding plant development. Numerous studies have utilized histological, surgical and genetic approaches to analyze the properties of the apical meristem, for example, J. I. Medford, Plant Cell 1992, 4, 1029-1039. The apical meristem is commonly described in terms of three generative tissue layers, S. Satina, et al., Am. J. Bot. 1940, 27, 895-905. The outer layer, the L1 layer, divides mostly in plane anticlinical to the surface of the apical meristem and gives rise to the shoot epidermis. Cells of L2 layer divide mainly in an anticlinical plane, but divide periclinically during primordia formation. Cells of the L3 layer divide in all planes and give rise to the central core of the plant.
Although the fate of cells produced within the apical meristem is clear, much remains to be learned about the regulatory events that occur during cell differentiation. For example, what processes define the differentiation of a cell from the L1, L2, or L3 layers as it emerges from the apical meristem and slowly develops into a fully differentiated shoot cell. In the case of the L1 layer, it is known that mature epidermal cells arise by successive anticlinical divisions from the protodermis of the apical meristem, but are there any specific molecular or cytological changes involved in the differentiation of cells arising from the apical meristem? As judged by light microscopy there is no clear visible demarcation between proto- and epidermal cells in pea, B. F. Thomson and P. M. Miller, Am. J. Bot. 1962, 49, 303-310. In fact, in pea, epidermal cells remain undifferentiated and indistinguishable from the internal tissue as far as five nodes from the apical meristem in 8 day old seedlings, Thomson and Miller, supra.
The plant epidermis represents the primary barrier and interactive surface with the environment (Esau, K, Anatomy of Seed Plants. John Wiley and Sons, Inc., New York, 1960). Molecules and structures within the epidermis are believed to play roles in defense from microbial or animal attack, attracting beneficial insects by the synthesis of pigments or volatile chemicals, mechanical support, and prevention of water loss. Since plants and plant pests have co-evolved over millions of years, a plant's natural defenses are often inadequate to protect it from microbial or animal attack. The introduction of foreign genes, cloned from plant, animal, and/or microbial sources, by genetic engineering, represents a strategy with which to enhance the defense properties of a plant. Examples of this strategy include transgenic tobacco plants protected from insect attack using the Bacillus thuringiensis endotoxin expressed under the control of .sup.35 S CaMV promoter (Vaeck et al., Nature 1987, 328, 33-37), and tobacco plants protected against fungal attack using a bean chitinase expressed under the control of the CaMV promoter (Broglie et al., Science 1991, 254, 1194-1197.
For optimal efficiency, the genetically engineered defense molecule should be highly expressed in the plant tissue first encountered by the pest which in most cases is the outer epidermis.
Maiti et al. (Maiti et al., Planta 1993, 190, 241-246) reported the characterization of mRNA sequences encoding a lectin-like protein, and corresponding cDNAs that accumulate in the shoot-apex epidermis of the garden pea (Pisum sativum). Others (Sterk et al., Plant Cell 1991, 3, 907-921) have reported the characterization of mRNA sequences encoding a lipid transfer protein gene that is highly expressed in epidermal cells of carrot shoot-apices. Clark et al. (Clark et al., Plant Cell 1992, 4, 1189-1198) reported the characterization of mRNA sequences of lipid transfer protein genes expressed in the epidermis of Pachyphytum.
Other examples of mRNA specific to the epidermis include reports by Schmelzer et al. (Schmelzer et al., Proc. Natl. Acad. Sci. USA 1988, 85, 2989-2993; Schmelzer et al., Plant Cell 1989, 1, 993-1001) who identified mRNAs encoding chalcone synthase (CHS) and phenyl-alanine ammonia lyase (PAL) gene families that accumulate in epidermal cells in response to chemical induction. In the absence of induction PAL and CHS mRNAs are expressed in parenchymotous mesophyll tissue as well.
Goodrich et al. (Goodrich et al., Cell 1992, 68, 955-964) reported that mRNAs encoding enzymes involved in anthocyanin biosynthesis in snapdragon flowers are expressed in specialized epidermal cells for limited periods during flower bud development. Wyatt et al. (Wyatt et al., Plant Cell 1992, 4, 99-110) reported that mRNAs encoding the soybean proline-rich cell wall proteins SbPRP1, SbPRP2, and SbPRP2 accumulate at certain developmental stages in the epidermis but are expressed at different times in the vascular tissue as well.
To demonstrate which portion of a gene is required to direct the pattern of tissue specific expression described above, it is necessary to isolate the putative regulatory region of a gene and test its ability to direct the expression of a reporter gene in transgenic plants. This may involve, for example, placing the promoter (5' upstream region) of a gene in combination with the coding region of a reporter gene, for example the bacterial gene .beta.-glucuronidase. Jefferson et al., EMBO J. 1987, 6, 3901-3907. .beta.-Glucuronidase activity can be readily assayed in situ using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic acid.
Promoters shown to direct tissue specific expression include those active in mesophyll and palisade of leaves (Broglie et al., Science 1984, 234, 838-845), dividing shoot and root tissues meristems (Atanassova et al., Plant J. 1992, 2, 291-300), pollen, (Guerrero et al., Mol. Gen. Genet. 1990, 224, 161-168), seed endosperm, (Stalberg et al., Plant Mol. Biol. 1993, 23, 671-683), root epidermis (Suzuki et al., Plant Mol. Biol 1993, 21, 109-119) and root meristems, vascular tissue and nodules (Bogusz et al., Plant Cell 1990, 2, 633-641). The literature contains relatively few reports of promoters that are expressed in the epidermis, this includes promoters active in the epidermal cells of flowers (Koes et al., Plant Cell 1990, 2, 379-392) and those are expressed in the epidermis in response to wounding (Liang et al., Proc. Natl. Acad. Sci. USA 1989, 86, 9284-9288). There are no known reports of promoters capable of directing expression to the epidermis of the growing shoot tip.